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Title: Use of Biomarker Modulation in Normal Mammary Epithelium as a Correlate for Efficacy

of Chemopreventive Agents Against Chemically-Induced Cancers

Authors: Ronald A. Lubet1, Brandy M. Heckman-Stoddard2, Jennifer T. Fox1, Fariba

Moeinpour3, M. Margaret Juliana3, Robert H. Shoemaker1, and Clinton J. Grubbs3

Affiliations: 1Chemopreventive Agent Development Research Group, Division of Cancer

Prevention, National Cancer Institute, Rockville, MD; 2Breast and Gynecologic Cancer

Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, MD;

3Department of Surgery, University of Alabama at Birmingham, Birmingham, AL

Running Title: Biomarker Modulation and Chemopreventive Efficacy

Corresponding Author: Ronald A. Lubet, National Cancer Institute, 9609 Medical Center Drive

Rockville, MD 20850. Phone (240) 276-5997, E-Mail: [email protected]

Conflict of Interest: The authors declare no conflict of interest

Cancer Research. on January 23, 2021. © 2019 American Association forcancerpreventionresearch.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 23, 2019; DOI: 10.1158/1940-6207.CAPR-19-0318

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ABSTRACT

In both estrogen receptor/progesterone receptor-positive (ER+/PR+) human breast cancer and in

ER+/PR+ cancers in the methylnitrosourea (MNU)-induced rat model, short-term modulation of

proliferation in early cancers predicts preventive/therapeutic efficacy. We determined the effects

of known effective/ineffective chemopreventive agents on proliferative index (PI) in both rat

mammary epithelium and small cancers. Female Sprague-Dawley rats were treated with MNU

at 50 days of age. Five days later, the rats were treated with the individual compounds for a

period of 14 days. At that time, normal mammary tissue from the inguinal gland area was

surgically removed. After removal, the rats remained on the agents for an additional 5 months.

This cancer prevention study confirmed our prior results of striking efficacy with tamoxifen,

vorozole, Targretin, and gefitinib, and no efficacy with metformin, naproxen, and Lipitor.

Employing a separate group of rats, the effects of short-term (7 days) drug exposure on small

palpable cancers were examined. The PI in both small mammary cancers and in normal

epithelium from control rats was >12%. In agreement with the cancer multiplicity data,

tamoxifen, vorozole, gefitinib, and Targretin all strongly inhibited proliferation (>65%) (P<0.025)

in the normal mammary epithelium. The ineffective agents metformin, naproxen, and Lipitor

minimally affected PI. In the small cancers, tamoxifen, vorozole and Targretin all reduced the PI,

while metformin and Lipitor failed to do so. Thus, short-term changes in the PI in either normal

mammary epithelium or small cancers correlated with long-term preventive efficacy in the MNU-

induced rat model.




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INTRODUCTION

The primary use of animal models in the field of chemoprevention is to screen for

potential agents that may be useful clinically. However, animal models can also be used to

examine surrogate biomarkers that correlate with efficacy. Modulation of surrogate biomarkers

is used in early phase prevention studies as a marker of efficacy. In breast cancer treatment

and prevention, these markers are often measured using a pre-surgical or neoadjuvant study in

which women diagnosed with early stage cancer or preneoplastic lesions are treated for a

limited length of time with an agent prior to initial surgery (1). The most common biomarkers

employed have been proliferation-related biomarkers such as Ki67 or PCNA. Two classes of

agents that have proven highly effective as preventive agents in Phase III clinical prevention

trials, selective estrogen receptor modulators (SERMs) and aromatase inhibitors (2,3), have

been shown to significantly reduce Ki67 in pre-surgical studies as well (4). Approximately 10

years ago, we confirmed that there was a strong correlation between the chemopreventive

efficacy of a variety of agents and the agent’s ability to inhibit proliferation in small palpable

mammary cancers following short-term treatment in the methylnitrosourea (MNU)-induced rat

model (5). One alternative approach to examining proliferative changes in tumors would be to

test the effects of agents on normal mammary epithelium. Although this approach has certain

technical challenges when performed in humans because Ki67 can be low in normal breast

epithelium, it has been used in women at high risk of developing breast cancer (6), and has

yielded positive results with both SERMs and aromatase inhibitors (7,8).

Rat models of breast cancer in which cancers are induced by the carcinogens

dimethylbenzanthracene (DMBA) or MNU have been employed for many decades (9). The

MNU-induced rat model of breast cancer induces estrogen receptor-positive (ER+) cancers that

are similar by array analyses to highly-differentiated ER+ human breast cancers (10). In

addition, these tumors respond both in a preventive setting and in a therapeutic setting to

hormonal treatments that modulate human ER+ cancers, including SERMs, aromatase inhibitors

and ovariectomy (11,12). Since we had previously identified a wide variety of highly effective

and ineffective cancer preventive agents in the MNU model, we examined the correlation

between short-term proliferative effects on normal mammary epithelium and long-term

chemopreventive efficacy. This study was facilitated by the fact that the inguinal mammary

tissue adjacent to the linea alba in young rats has a high concentration of terminal end buds and

ducts, and that the proliferative rates in these tissues are quite high. Because of these

characteristics, we were able to determine the proliferative index (PI) in normal epithelium after

14 days of treatment with the various agents. In brief, we tested a variety of compounds that




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were known to be effective or ineffective preventive agents based on our prior published data

(12-15). Here we sought to correlate the short-term change in proliferation in “high risk”

mammary epithelium with the short-term change seen in mammary tumors and the effect of the

agent on tumor incidence and multiplicity.

MATERIALS AND METHODS Chemicals and animals

MNU was obtained from the NCI Chemical Carcinogen Repository. Teklad diet and

female Sprague-Dawley rats were obtained from Envigo, Inc., Indianapolis, IN. Gefitinib,

Targretin, vorozole, metformin and Lipitor were supplied by the NCI Cancer Prevention

Repository. Naproxen and tamoxifen were purchased from Sigma Chemical Co., St. Louis, MO.

Gefitinib (10 mg/kg BW), vorozole (1.25 mg/kg BW) and metformin (150 mg/kg BW) were

administered by gavage (0.5 ml/gavage) on a daily basis. The vehicle for vorozole and gefitinib

was ethanol: polyethylene glycol 400 (10:90; v/v), while metformin was administered in saline.

Tamoxifen (3.3 mg/kg of diet), Targretin (150 mg/kg of diet), naproxen (400 mg/kg of diet) and

Lipitor (150 mg/kg of diet) were administered in the diet. The agents were incorporated into the

feed (Teklad, 4% fat) using a Patterson-Kelly blender with intensifier bar. Fresh diet was

provided to the rats 3x/week.

Prevention studies with various agents

All animal experiments were conducted in AAALAC-approved facilities following

procedures approved by the Institutional Animal Care and Use Committee at the University of

Alabama at Birmingham. Treatment of the female Sprague-Dawley rats was as previously

described (12,14). In brief, rats were injected IV with MNU (75 mg/kg BW) via the jugular vein at

50 days of age. Treatment of rats with the various agents by gavage or in the diet was initiated

five days after MNU administration (or at 55 days of age). The number of rats/group was 20.

We had previously determined effective daily doses for the various agents (12-15). These doses

were all less than or equal to the daily human equivalent dose (HED) based on FDA scaling,

with the exception of Lipitor which was a slightly higher dose. Two weeks after the initiation of

treatment with the various agents, mammary tissue was obtained from the inguinal mammary

glands as described below. Treatment with the agents continued for approximately 5 months.

The rats were palpated twice a week for the development of mammary tumors. At the end of

the study, tumors were weighed and submitted for histological evaluation. In all studies, rats

were weighed 1x/week. The body weights of the control and treated rats did not differ by more

than 5% in any of the long-term prevention studies.




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To determine proliferative effects in normal epithelium, the rats in the prevention study

underwent surgery 14 days after treatment initiation to remove mammary tissue from the

inguinal glands. Survival surgery was performed in which rats were anesthetized with

isoflurane. Mammary tissue was excised from an area in the inguinal glands (adjacent to the

linea alba) which has a high concentration of epithelial cells. Tissues were fixed in 10% formalin

for 24 hours and then transferred to 70% ethanol at room temperature. Tissues were then

embedded in paraffin blocks and sections cut (4 microns thick).

To determine proliferative effects in small cancers, a separate group of rats (5/group)

received MNU at 50 days of age and were palpated 2x/week for the appearance of mammary

cancers. When an animal developed a cancer of approximately 100-200 mm2, the rat received

the indicated agent at the dose specified above for 7 days. One day after the last treatment

with the agents, the animals were sacrificed, and the mammary cancers were excised. The

harvested tumors were fixed in 10% formalin for 24 hours at room temperature and were then

transferred to 70% ethanol until histologically processed. Tissues were then embedded in

paraffin blocks and sections cut (4 microns thick).

Immunohistochemistry

The mammary gland and tumor tissues from 10 and 5 rats/group, respectively, were de-

paraffinized with xylene and placed in ethanol. Antigen retrieval employed boiling in sodium

citrate (pH, 6.0) for 20 minutes. Slides were then covered with peroxidase block for 3 hours

and washed with tris buffer. The tissues were incubated with Ki67 primary antibody (Abcam,

Cambridge, MA) for one hour at room temperature. Processing and staining of tissue were

performed according to the manufacturer's procedures (DAKA Envision + Kits, Carpin Teria,

CA). Tissues were then washed and dehydrated in ethanol and xylene. The images were

captured and counted using the Aperio Scan Scope imaging system (Aperio Imaging, Visa,

CA). For counting the cells, each area containing mammary ductal epithelial cells was

randomly analyzed (stained cells ÷ total cells counted) by a program within Scan Scope.

Approximately 15 areas/slide were analyzed to ensure that a minimum of 1000 cells/tissue were

counted.

Statistical analysis

Final mammary cancer multiplicities and weights were compared statistically using a

non-parametric Mann-Whitney rank analysis test since the data does not follow a “normal”

curve. Proliferation indices are presented as mean ± standard error and were compared




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statistically using one-way ANOVA. Correlation coefficients were calculated using Microsoft

Excel. P<0.05 was used to determine a statistically significant difference.

RESULTS

Chemopreventive effects of various agents

We had previously identified a variety of agents that were either highly effective

(tamoxifen, vorozole, Targretin and gefitinib) or ineffective (metformin, Lipitor and naproxen) in

long-term prevention studies in the MNU rat model of mammary cancer (12-16) (Table 1). We

had also previously shown that tamoxifen, vorozole, and Targretin reduced both cancer

multiplicity and PI, and increased apoptotic index in a dose-dependent manner in a short-term

study in the same animal model (5) (Table S1). To determine whether there was a correlation

between the long-term efficacy of these agents and their short-term effects on cellular

proliferation in normal epithelium, we repeated these chemoprevention studies in the MNU rat

model, taking biopsies of normal mammary tissue from the inguinal gland 14 days after initiating

treatment with the agents. After removal of mammary tissue from the inguinal gland, drug

treatment of rats continued for another 5 months. Consistent with our previous findings,

tamoxifen, vorozole, gefitinib and Targretin were profoundly effective in reducing cancer

development and final tumor weight; and naproxen and metformin both significantly increased

cancer multiplicity and final tumor weights. Lipitor had no statistically significant effect (Figures

1A and 1B; Tables 2 and S2).

Effects of preventive agents on the PI in normal mammary epithelium and correlation with long-term preventive effects

To correlate these efficacy data with the PI in the normal mammary epithelium, we

stained the biopsied tissue samples collected 14 days after treatment initiation for Ki67

expression (Figure 2A). A minimum of 1000 cells/tissue were counted using the Scan-scope

instrument described above. The normal epithelium from the mammary gland of control rats had

a relatively high number of proliferating cells that approached the values observed in cancers.

This PI was significantly (P<0.05) reduced (ANOVA) by all of the agents that showed

chemopreventive efficacy; vorozole and Targretin both reduced the PI in normal epithelium by

>80%, while tamoxifen and gefitinib reduced the PI by approximately 75% and 60%,

respectively (Figure 2B). The two agents that did not demonstrate long-term chemopreventive

efficacy (naproxen and metformin) marginally altered the PI in normal epithelium, respectively

(Figure 2B); neither effect was statistically significant. Lipitor, which reduced tumor multiplicity




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by 17%, reduced the PI by approximately 35%, although this reduction failed to achieve

statistical significance (P>0.05) (Figure 2B). The correlation between the effects of short-term

treatment on the proliferation of normal mammary epithelium and the long-term effects on

cancer multiplicity was strong, with a Pearson correlation coefficient (R) of 0.87 and a coefficient

of determination (R2) of 0.76 (P<0.01) (Figure 2C).

Effects of preventive agents on the PI following the short-term treatment of small cancers and correlation with long-term preventive effects

To correlate the long-term efficacy data with the PI in mammary cancers, we injected a

separate group of rats with MNU and let individual animals develop small palpable mammary

cancers before treating them with the various agents for 7 days. The cancers were then

collected from these animals and stained for Ki67 expression (Figure 3A). As observed in the

normal epithelium, the effective chemopreventive agents (vorozole, tamoxifen and Targretin)

significantly reduced proliferation in the tumors by approximately 70-80% (P<0.05) (Figure 3B).

Consistent with their failure to reduce cancer multiplicity, neither Lipitor not metformin

significantly altered the PI (Figure 3B). Again, the correlation between the effects of short-term

treatment on the proliferation of small palpable cancers and the long-term effects on cancer

multiplicity was strong, with an R value of 0.97 and an R2 value of 0.95 (P<0.01) (Figure 3C).

DISCUSSION One of the primary objectives of preclinical models is to identify surrogate endpoints that

can be used in early phase prevention trials that are predictive of cancer preventive efficacy in

Phase III trials. Phase II trials have often employed Ki67 as the biomarker of interest. In the

area of breast cancer, the two classes of agents that have proven effective in large Phase III

prevention clinical trials are the SERMs (tamoxifen, raloxifene) and the aromatase inhibitors

(letrozole and exemestane) (2,3). Both classes of agents have been shown to significantly

reduce Ki67 in normal mammary epithelium in humans (7,8), and have proven to be highly

effective in reducing the proliferation of breast cancer cells in a pre-surgical trials (4). This

efficacy in early tumors is not surprising since these classes of agents are clinically effective

therapies in ER+/PR+ breast cancer. Here, we sought to investigate the correlation between

long-term chemopreventive efficacy and short-term PI in normal mammary epithelium and

palpable cancers in the MNU-induced rat model using a series of agents that are known to be

effective or ineffective in this model. The question arises whether one might use another

biomarker either in addition to or instead of Ki67. In fact, in a recent study with the aromatase




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inhibitor vorozole, we employed a variety of proliferation-related proteins as potential biomarkers

in both normal epithelia and in tumor lesions (17). The reason we have not employed any of

these in the present studies is that they are not standardized or virtually ever employed

clinically. Thus, there is no data to tell us whether comparable results can be obtained in the

human. In contrast, for Ki67, there is significant human data available for comparison.

In agreement with prior studies from our laboratory and others (11,12), and consistent

with the clinical trials discussed above, we demonstrated that tamoxifen and vorozole are highly

effective in reducing mammary cancer multiplicity and weight in the ER+ model. It was found

that short-term treatment with these agents also reduced proliferation in the inguinal mammary

gland as well as in small palpable cancers. Because the normal mammary epithelium and

cancers of this model are both ER+, it is not surprising that a SERM (tamoxifen) and an

aromatase inhibitor (vorozole) were highly effective in reducing the PI in both.

In addition to testing tamoxifen and vorozole, we confirmed our previous results (13,14)

demonstrating that gefitinib and Targretin are highly effective as preventive agents in the MNU

model. Chemopreventive activity correlated well with the PI in both the normal mammary

epithelium and in cancers, despite the fact that neither gefitinib (an EGFR inhibitor) nor

Targretin (an RXR agonist) directly target the hormonal axis. Interestingly, two clinical studies

have shown the efficacy of EGFR inhibitors against ER+/ PR+ breast cancers in women by

employing either proliferation (18,19) or therapeutic efficacy in a neoadjuvant setting (20). In

prior studies, we have also shown that one can achieve a dose-dependent response to with

either vorozole (12) or wide variety of other agents (5), when examining proliferation in lesions.

We summarize some of this data in the supplement (Table S1). In contrast to tamoxifen,

vorozole, gefitinib and Targretin, the agents naproxen, metformin, and Lipitor showed no

significant chemopreventive effects in the long-term cancer study, and likewise did not

significantly reduce the PI of normal mammary epithelium or small cancers in the short-term

biomarker study. In fact, naproxen and metformin both increased tumor multiplicity. However,

we have routinely not given emphasis to the tumor-promoting effects of agents in the MNU

model since the studies are performed in carcinogen-treated Sprague-Dawley rats, whereas

carcinogenesis assays performed by the National Toxicology Program use rodents that have

not previously been exposed to a carcinogen.

The primary objective of this paper was to examine the relationship between short-term

effects on Ki67 in the normal epithelium and small palpable tumors with long-term

chemopreventive activity in the MNU breast cancer model. However, there is at least some

basis for comparing the Ki67 results in rats and humans. Examining Ki67 in lesions, we




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observed an approximately 70-80% reduction in proliferation in both the normal rat epithelium

and in tumors following treatment with either tamoxifen or an aromatase inhibitor. These results

parallel those observed following the similar treatment of ER+/PR+ human tumors in a

neoadjuvant setting (4) and in two studies with normal epithelium and fine needle aspirates

(6,7). Furthermore, the EGFR inhibitor erlotinib showed striking inhibition of proliferation in

ER+/PR+ tumors (18), which is similar to our present data in normal rat epithelium and parallels

our prior data in rat tumors (5,14). Whereas our own Ki67 data with metformin in either tumors

or normal epithelium was negative corresponding to our prevention results, Ki67 data in humans

have been more mixed. Four trials have examined the short-term effects (1–4 weeks) of various

doses of metformin on cell proliferation (as assessed by the expression of Ki-67) in tissues from

women awaiting surgery for breast cancer (pre-surgical trials) (21-24). In the largest

randomised, double-blind, placebo-controlled study of the effect of metformin on Ki67 in breast

cancer, the change in Ki67 between diagnostic biopsy and surgical specimen was not significant

relative to placebo (21). However, women with higher insulin resistance (HOMA-IR > 2.8) had a

non-significant 10.5% decrease in Ki67. While a recently completed study in a majority

Hispanic population with a historical control group matched for age, BMI and stage showed no

reduction in Ki67 in the metformin arm or in the untreated control group (24) , two single-arm

trials (metformin baseline vs pre-surgery) (22,23) resulted in limited, but statistically significant,

decreases in proliferation (<10%). Thus, none of the studies yielded the striking inhibitions

achieved by tamoxifen, anastrozole or erlotinib in humans (4,7,8,18).

The results presented in this manuscript confirm the reproducibility of our data with

respect to both effective and ineffective cancer chemopreventive agents. More importantly, they

show that there is a strong correlation between a reduced PI in normal epithelium in the

mammary gland or in palpable lesions and long-term cancer preventive activity with a variety of

agents. This supports the use of this biomarker in prevention trials. However, when considering

the translation of our data with normal epithelium to human trials, there are several points that

should be noted. First, the mammary gland biopsied from a 70 day-old rat has a high

concentration of terminal end buds, resulting in a high concentration (approximately 15%) of

normal epithelial cells in the gland. This is much higher than the concentration of normal

epithelial cells observed in mammary glands of a mature rat, and (more importantly) much

higher than that in adult humans where epithelial cells may represent only ≤3% of the cells

obtained following a fine-needle aspirate of a normal gland. Second, because 70-day-old rats

still have developing mammary glands, approximately 12-16% of epithelial cells in the inguinal

mammary gland are proliferating based on Ki67 staining. Thus, the PI in the normal mammary




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epithelium of these rats is almost as high as that in the mammary cancers produced in this

model, and is approximately equal to the PI in human ER+/PR+/Neu- breast cancers (4). In adult

women, the mammary gland has both a low incidence of epithelial cells and a low PI. As a

result, most of the measurements of proliferation in humans are determined in lesions

(hyperplasias, DCIS or early tumors). The final limitation of using normal epithelium involves

loss of specificity of agents. For example, SERMS and aromatase inhibitors are generally

effective with tumors that are ER+/ PR+/Neu-, but they are much less effective against ER+/

PR+/Neu+ cancers (4). We have employed small tumors in the MNU model to examine the

efficacy of the farnesyl transferase inhibitor Tipifarnitib (25,26) and found that lesions with an

HRAS mutation are more susceptible than tumors without these mutations. This specific

approach involving sequencing of mutated genes or array analysis can only be performed in

clear lesions, not in normal epithelium. However, despite these challenging hurdles, this study

showed it is possible to biopsy animals to determine the correlation between surrogate

biomarkers and tumor endpoints. Ki67, which is used in many Phase II studies, is a reasonable

surrogate marker based on these studies.

ACKNOWLEDGEMENTS

The work on this manuscript was supported by the National Cancer Institute (Contract

HHSN261201200021I, Task Order HHSN26100003 to C. Grubbs).

REFERENCES

1. Klintman M, Dowsett M. Early Surrogate Markers of Treatment Activity: Where Are We Now? J Natl Cancer Inst Monogr 2015;2015(51):24-8 doi 10.1093/jncimonographs/lgv002.

2. Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998;90(18):1371-88.

3. Goss PE, Ingle JN, Ales-Martinez JE, Cheung AM, Chlebowski RT, Wactawski-Wende J, et al. Exemestane for breast-cancer prevention in postmenopausal women. N Engl J Med 2011;364(25):2381-91 doi 10.1056/NEJMoa1103507.

4. Dowsett M, Smith IE, Ebbs SR, Dixon JM, Skene A, Griffith C, et al. Short-term changes in Ki-67 during neoadjuvant treatment of primary breast cancer with anastrozole or tamoxifen alone or combined correlate with recurrence-free survival. Clin Cancer Res 2005;11(2 Pt 2):951s-8s.

5. Christov K, Grubbs CJ, Shilkaitis A, Juliana MM, Lubet RA. Short-term modulation of cell proliferation and apoptosis and preventive/therapeutic efficacy of various agents in a mammary cancer model. Clin Cancer Res 2007;13(18 Pt 1):5488-96 doi 10.1158/1078-0432.CCR-07-0404.




11

6. Fabian CJ, Kimler BF, Elledge RM, Grizzle WE, Beenken SW, Ward JH. Models for early chemoprevention trials in breast cancer. Hematol Oncol Clin North Am 1998;12(5):993-1017.

7. Fabian CJ, Kimler BF, Zalles CM, Khan QJ, Mayo MS, Phillips TA, et al. Reduction in proliferation with six months of letrozole in women on hormone replacement therapy. Breast Cancer Res Treat 2007;106(1):75-84 doi 10.1007/s10549-006-9476-5.

8. Fabian CJ, Kimler BF, Zalles CM, Phillips TA, Metheny T, Petroff BK, et al. Clinical Trial of Acolbifene in Premenopausal Women at High Risk for Breast Cancer. Cancer Prev Res (Phila) 2015;8(12):1146-55 doi 10.1158/1940-6207.CAPR-15-0109.

9. Huggins CB, Ueda N, Wiessler M. N-Nitroso-N-methylurea elicits mammary cancer in resistant and sensitive rat strains. Proc Natl Acad Sci U S A 1981;78(2):1185-8.

10. Chan MM, Lu X, Merchant FM, Iglehart JD, Miron PL. Gene expression profiling of NMU-induced rat mammary tumors: cross species comparison with human breast cancer. Carcinogenesis 2005;26(8):1343-53 doi 10.1093/carcin/bgi100.

11. Gottardis MM, Jordan VC. Antitumor actions of keoxifene and tamoxifen in the N-nitrosomethylurea-induced rat mammary carcinoma model. Cancer Res 1987;47(15):4020-4.

12. Lubet RA, Steele VE, DeCoster R, Bowden C, You M, Juliana MM, et al. Chemopreventive effects of the aromatase inhibitor vorozole (R 83842) in the methylnitrosourea-induced mammary cancer model. Carcinogenesis 1998;19(8):1345-51.

13. Lubet RA, Christov K, Nunez NP, Hursting SD, Steele VE, Juliana MM, et al. Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis. Carcinogenesis 2005;26(2):441-8 doi 10.1093/carcin/bgh338.

14. Lubet RA, Szabo E, Christov K, Bode AM, Ericson ME, Steele VE, et al. Effects of gefitinib (Iressa) on mammary cancers: preventive studies with varied dosages, combinations with vorozole or targretin, and biomarker changes. Mol Cancer Ther 2008;7(4):972-9 doi 10.1158/1535-7163.MCT-07-2141.

15. Thompson MD, Grubbs CJ, Bode AM, Reid JM, McGovern R, Bernard PS, et al. Lack of effect of metformin on mammary carcinogenesis in nondiabetic rat and mouse models. Cancer Prev Res (Phila) 2015;8(3):231-9 doi 10.1158/1940-6207.CAPR-14-0181-T.

16. Lubet RA, Steele VE, Shoemaker RH, Grubbs CJ. Screening of Chemopreventive Agents in Animal Models: Results on Reproducibility, Agents of a Given Class, and Agents Tested During Tumor Progression. Cancer Prev Res 2018;11(10):595-605 doi 10.1158/1940-6207.Capr-18-0084.

17. Lu Y, You M, Ghazoui Z, Liu P, Vedell PT, Wen W, et al. Concordant effects of aromatase inhibitors on gene expression in ER+ Rat and human mammary cancers and modulation of the proteins coded by these genes. Cancer Prev Res (Phila) 2013;6(11):1151-61 doi 10.1158/1940-6207.CAPR-13-0126.

18. Baselga J, Albanell J, Ruiz A, Lluch A, Gascon P, Guillem V, et al. Phase II and tumor pharmacodynamic study of gefitinib in patients with advanced breast cancer. J Clin Oncol 2005;23(23):5323-33 doi 10.1200/JCO.2005.08.326.

19. Guix M, Granja Nde M, Meszoely I, Adkins TB, Wieman BM, Frierson KE, et al. Short preoperative treatment with erlotinib inhibits tumor cell proliferation in hormone receptor-positive breast cancers. J Clin Oncol 2008;26(6):897-906 doi 10.1200/JCO.2007.13.5939.

20. Polychronis A, Sinnett HD, Hadjiminas D, Singhal H, Mansi JL, Shivapatham D, et al. Preoperative gefitinib versus gefitinib and anastrozole in postmenopausal patients with oestrogen-receptor positive and epidermal-growth-factor-receptor-positive primary




12

breast cancer: a double-blind placebo-controlled phase II randomised trial. Lancet Oncol 2005;6(6):383-91 doi 10.1016/S1470-2045(05)70176-5.

21. Bonanni B, Puntoni M, Cazzaniga M, Pruneri G, Serrano D, Guerrieri-Gonzaga A, et al. Dual effect of metformin on breast cancer proliferation in a randomized presurgical trial. J Clin Oncol 2012;30(21):2593-600 doi 10.1200/JCO.2011.39.3769.

22. DeCensi A, Puntoni M, Gandini S, Guerrieri-Gonzaga A, Johansson HA, Cazzaniga M, et al. Differential effects of metformin on breast cancer proliferation according to markers of insulin resistance and tumor subtype in a randomized presurgical trial. Breast Cancer Res Treat 2014;148(1):81-90 doi 10.1007/s10549-014-3141-1.

23. Hadad SM, Coates P, Jordan LB, Dowling RJ, Chang MC, Done SJ, et al. Evidence for biological effects of metformin in operable breast cancer: biomarker analysis in a pre-operative window of opportunity randomized trial. Breast Cancer Res Treat 2015;150(1):149-55 doi 10.1007/s10549-015-3307-5.

24. Kalinsky K, Crew KD, Refice S, Xiao T, Wang A, Feldman SM, et al. Presurgical trial of metformin in overweight and obese patients with newly diagnosed breast cancer. Cancer Invest 2014;32(4):150-7 doi 10.3109/07357907.2014.889706.

25. Lubet RA, Christov K, You M, Yao R, Steele VE, End DW, et al. Effects of the farnesyl transferase inhibitor R115777 (Zarnestra) on mammary carcinogenesis: prevention, therapy, and role of HaRas mutations. Mol Cancer Ther 2006;5(4):1073-8 doi 10.1158/1535-7163.MCT-05-0398.

26. Yao R, Wang Y, Lu Y, Lemon WJ, End DW, Grubbs CJ, et al. Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets. Carcinogenesis 2006;27(7):1420-31 doi 10.1093/carcin/bgi341.

TABLES

Table 1: Description of the agents used in these studies

Agent Description

Chemopreventive Activity in Prior

Mammary Cancer Studies (12-15)?

Tamoxifen

Selective estrogen receptor modulator (SERM) that binds estrogen receptor (ER) alpha and inhibits the stimulatory effects of estrogen in ER+ breast cancers. Has both therapeutic and preventive activity clinically. The dose employed in these studies is at the human equivalent dose (HED).

Yes

Vorozole

Small molecule competitive inhibitor of aromatase (CYP 27) that is similar to clinically-employed letrozole and anastrozole. Aromatase inhibitors (letrozole, exemustane) are used clinically in therapy and are effective in clinical prevention studies.

Yes

Targretin Pure RXR agonist employed clinically in the treatment of Cutaneous T cell Lymphoma (CTCL). The dose used in these studies is less than the HED for CTCL.

Yes

Gefitinib Small molecule competitive inhibitor of EGFR1. Effective in human ER+ tumors and effective in MNU model at roughly ½ of the HED.

Yes

Metformin Used to control blood sugar levels in Type 2 diabetics. Its No




13

mechanism of action is unclear. The dose employed in these studies is approximately equivalent to the commonly-employed human dose of 1500 mg.

Lipitor Competitive HMG CoA reductase inhibitor used to lower cholesterol levels. The dose employed in these studies is approximately 2-4X higher than that typically used in humans.

No

Naproxen

Non-steroidal anti-inflammatory drug (NSAID) that competitively inhibits COX 1/2. It is a highly effective chemopreventive agent in colon and bladder cancer models. The dose employed in these studies is approximately equivalent to 350 mg in humans.

No

Table 2: Effect of various agents on final mammary cancer multiplicity and weight.

Agent Multiplicity (# of Cancers/Rat)* Cancer Weight (g)

Control 2.58 4.58

Vorozole 0.25 (90% decrease, p=1e-5) 0.36 (92% decrease, p=6e-5)

Targretin 0.20 (92% decrease, p=6e-6) 0.30 (93% decrease, p=2e-5)

Lipitor 2.15 (17% decrease, p=0.6) 5.11 (12% increase, p=0.7)

Control 2.90 6.79

Naproxen 4.20 (45% increase, p=0.02) 10.49 (54% increase, p=0.05)

Gefitinib 0.20 (93% decrease, p=2e-6) 0.14 (98% decrease, p=1e-6)

Tamoxifen 0.00 (100% decrease, p=1e-9) 0.00 (100% decrease, p=1e-7)

Metformin 4.95 (71% increase, p=0.01) 9.71 (43% increase, p=0.33)

*Based on histopathological analysis following animal sacrifice; p values were calculated using the Mann-Whitney test.

FIGURE LEGENDS

Figure 1. Effect of various agents on the appearance of palpable mammary cancers in MNU-

treated rats. Five days after MNU injection, rats (20/group) were treated daily with (A) vorozole

(1.25 mg/kg BW by gavage), Targretin (150 mg/kg of diet), or Lipitor (150 mg/kg of diet). In a

separate study (B), the rats received naproxen (400 mg/kg of diet), gefitinib (10 mg/kg BW by

gavage), tamoxifen (3.3 mg/kg of diet), or metformin (150 mg/kg BW by gavage). The rats were

palpated twice a week for the development of mammary cancers.

Figure 2. Effect of various agents on the PI of normal mammary epithelium from MNU-treated

rats. Fourteen days after the initiation of treatment with the agents, mammary tissue was

removed from the inguinal mammary glands of the MNU-treated rats in Figure 1, and stained for

epithelial cell proliferation. (A) Representative images of Ki67 staining are shown. (B) The PI

was determined by calculating the percentage of Ki67 positive cells; a minimum of 1000




14

cells/tissue were counted. The data represent the average from 10 rats/group + standard error.

The (*) indicates statistical significance (P<0.05) as determined by ANOVA. (C) The average

PIs in the normal mammary epithelium (NE) were normalized to their respective controls

(average PI of control = 1.0) and were plotted as a function of the normalized average cancer

multiplicities (average multiplicity of control = 1.0) reported in Table 2. The Pearson correlation

coefficient (R) and coefficient of determination (R2) are shown on the graph.

Figure 3. Effect of various agents on the PI in small palpable cancers. A separate group of

MNU-treated rats (5/group) were allowed to develop mammary cancers. When an animal

developed a cancer of approximately 100-200 mm2, the rat was treated with vorozole (1.25

mg/kg BW by gavage), Targretin (150 mg/kg of diet), Lipitor (150 mg/kg of diet), tamoxifen (3.3

mg/kg of diet), or metformin (150 mg/kg BW by gavage) for 7 days. Upon termination of the

experiment, the cancers were removed and stained for Ki67. (A) Representative images of Ki67

staining are shown. (B) The PI was determined by calculating the percentage of Ki67-positive

cells; a minimum of 1000 cells/tissue were counted. The data represent the average from 5

rats/group + standard error. The (*) indicates statistical significance (P<0.05) as determined by

ANOVA. (C) The average PIs in the cancers were normalized to the control (average PI of

control = 1.0) and were plotted as a function of the normalized average cancer multiplicities

(average multiplicity of control = 1.0) reported in Table 2. The Pearson correlation coefficient (R)

and coefficient of determination (R2) are shown on the graph.













Published OnlineFirst December 23, 2019.Cancer Prev Res Ronald A Lubet, Brandy M. Heckman-Stoddard, Jennifer T Fox, et al. Chemically-Induced Cancersa Correlate for Efficacy of Chemopreventive Agents Against Use of Biomarker Modulation in Normal Mammary Epithelium as

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